Novel oxidase gene and method for producing 3-indole-pyruvic acid by utilizing the gene

ABSTRACT

It is an object of the present invention to provide a procedure for realizing inexpensive and simple production of 3-indole-pyruvic acid. A transformant is made using a polynucleotide having a specific nucleotide sequence encoding a protein having an oxidase activity, and oxidase is generated by culturing the transformant in a medium to accumulate the oxidase in the medium and/or the transformant. Further, tryptophan is converted into 3-indole-pyruvic acid in the presence of the transformant and/or a culture thereof to produce 3-indole-pyruvic acid.

TECHNICAL FIELD

The present invention relates to a novel oxidase gene and a novel methodfor producing 3-indole-pyruvic acid utilizing the gene.

BACKGROUND ART

In a chemical method for producing 3-indole-pyruvic acid by Giovanna DeLuca et al., 3-indole-pyruvic acid with a yield of 50 to 62% has beenobtained by reacting tryptophan as a starting material with pyridinealdehyde in the presence of a base for dehydrating a proton acceptor(see JP Sho-62-501912 [Patent Document 1], International Publication87/00169 Pamphlet [Patent Document 2]). In this method, the requiredbase and pyridine aldehyde are expensive and the yield is low. As aresult, its production cost seems to be very high. Politi Vincenzo etal. has obtained 3-indole-pyruvic acid with the yield of 64% by acondensation reaction using indole and ethyl-3-bromopyruvate ester oximeas raw materials followed by being subjected to an acid hydrolysis (seeEurope Patent Application Publication No. 421946 [Patent Document 3]).In this method, a purification step using silica gel is required, theyield is low, the raw materials are expensive and the cost is very highin the industrial production.

On the other hand, the method of using aminotransferase is known (seethe following reaction formula 1) as an enzymatic method for producing3-indole-pyruvic acid.

The method in which 3-indole-pyruvic acid is generated from 40 mML-tryptophan (L-Trp) and 80 mM 2-ketoglutaric acid by allowingL-tryptophan aminotransferase derived from Candida maltosa to act uponL-Trp and purified by an ion exchange resin to achieve the yield of 72%(see Bobe Ruediger et al., East Germany Patent DD 297190 [PatentDocument 4]), and the method in which aspartate aminotransferase isallowed to act upon L-Trp and 2-ketoglutaric acid to generate3-indole-pyruvic acid, which is then purified by extracting the reactionsolution with petroleum ether and fractionating by column chromatographyand the fraction is collected (see Mario Materazzi et al., JPSho-59-95894-A [Patent Document 5]) have been reported.Aminotransferases encoded by an aspC gene and a tyrB gene derived fromEscherichia coli is described in International Publication No.2003/091396 Pamphlet [Patent Document 6] and US Patent ApplicationPublication No. 2005/0282260 [Patent Document 7]. In these methods ofusing aminotransferase, the yield is low, keto acid such as2-ketoglutaric acid as an amino group acceptor is required as the rawmaterial in addition to L-Trp, and further an amino acid correspondingto the amino group acceptor present in an amount of equivalent moles to3-indole-pyruvic acid to be generated is produced as a byproduct.Furthermore, an excessive amount of keto acid relative to L-Trp is addedto the reaction system in order to enhance the yield. Thus, unreactedketo acid remains after the reaction. Due to these reasons, thepurification step using the ion exchange resin is required in order tocollect the objective 3-indole-pyruvic acid from the reaction solution.Thus, the manipulation is complicated and the cost is high.

The method of using L-amino acid oxidase is also known as the method forproducing 3-indole-pyruvic acid from L-Trp. In this regard, however,3-indole-pyruvic acid is decomposed into indoleacetic acid (see thefollowing reaction formula 3) by hydrogen peroxide produced as thebyproduct when tryptophan is oxidized by L-amino acid oxidase (see thefollowing reaction formula 2). Thus, the method of decomposing hydrogenperoxide by adding catalase to the reaction system (see the followingreaction formula 4) is proposed (see U.S. Pat. No. 5,002,963 to De Luca,et al., 1991 [Patent Document 8]).

In the above method, using an immobilized enzyme column in which L-aminoacid oxidase derived from a snake venom and catalase derived from bovineliver have been immobilized to a carrier, a solution containing L-Trp ispassed through the column to react, and produced 3-indole-pyruvic acidis adsorbed to an ion exchange column, eluted with methanol,subsequently exsiccated and collected. However, in this method, only 0.2g of 3-indole-pyruvic acid is acquired from starting 0.5 g of L-Trp, andthe yield is 40% that is low. Further, in this method, the steps such asimmobilizing the enzymes and purifying by the ion exchange resin arecomplicated, it is also necessary to collect or recycle unreacted L-Trp,and thus the cost is high.

Concerning L-amino acid oxidase derived from microorganisms, John A.Duerre et al. crudely purified L-amino acid oxidase (deaminase) derivedfrom Proteus rettgeri and detected an oxidation activity for L-Trp by anactivity measurement method of detecting an amount of consumed oxygen(see Journal of Bacteriology, 1975, vol. 121, No. 2, p 656-663[Nonpatent Document 1]). Furuyama et al. confirmed that L-phenylalanineoxidase derived from Pseudomonas sp. P-501 also acted upon L-Trp by theactivity measurement method of detecting the amount of consumed oxygen(see Noda Institute for Scientific Research, JP Sho-57-146573-A [PatentDocument 9]).

However, in any of these methods, the oxidase activity was detected bymeasuring the amount of consumed tryptophan, the amount of consumedoxygen or the amount of produced hydrogen peroxide, and indole-pyruvicacid was not quantified directly. This seems to be because3-indole-pyruvic acid is decomposed into indoleacetic acid by hydrogenperoxide produced by the reaction with amino acid oxidase. On the otherhand, there is no example in which 3-indole-pyruvic acid is generatedusing a microbial cell or a treated microbial cell, and how tryptophanis metabolized by the microorganism and what metabolite is generated bythe microorganism are unknown.

The microorganisms having the oxidase activity and belonging to generaAchromobacter, Proteus, Morganella, Pseudomonas and Neurospora aredisclosed in International Publication No. 03/056026 Pamphlet (patentDocument 10). However, when 3-indole-pyruvic acid was industriallyproduced on a large scale, there was a limit to produce it by amicrobial cell reaction alone.

Further, in the method of using aminotransferase or the method of usingL-amino acid oxidase derived from the snake venom among theaforementioned technology known publicly, the reaction yield is low,keto acid as the byproduct or unreacted L-tryptophan remains and ismixed in the reaction solution. Thus, a chromatographic separation stepis required for collecting 3-indole-pyruvic acid, thus the manipulationis complicated and the cost is high.

Under the circumstance as above, it is required to develop the methodfor producing 3-indole-pyruvic acid inexpensively and conveniently.

Patent Document 1: JP Sho-62-501912-A

Patent Document 2: International Publication WO87/00169 Pamphlet

Patent Document 3: Europe Patent Application Publication No. 421946

Patent Document 4: East Germany Patent DD 297190

Patent Document 5: JP Sho-59-95894-A

Patent Document 6: International Publication No. WO2003/091396 Pamphlet

Patent Document 7: US Patent Application Publication No. 2005/0282260

Patent Document 8: U.S. Pat. No. 5,002,963

Patent Document 9: JP Sho-57-146573-A

Patent Document 10: International Publication No. 03/056026 Pamphlet

Nonpatent Document 1: Journal of Bacteriology, 1975, vol. 121, No. 2, p656-663.

DISCLOSURE OF INVENTION Problem to be Solved by the Invention

3-Indole-pyruvic acid is extremely useful as a synthetic intermediate ofvarious pharmaceuticals and drinks and foods, particularly sweeteners.It is an object of the present invention to provide an inexpensive andsimple procedure for realizing the production of 3-indole-pyruvic acid.

Means for Solving Problem

As a result of an extensive study in the light of the abovecircumstance, the present inventors have found that 3-indole-pyruvicacid is generated and can be collected by cloning a novel oxidase genefrom a microorganism having an oxidase activity to construct atransformant and reacting tryptophan in the presence of the transformantand that preferably a reactivity is further enhanced by constructing thetransformant as a microbial strain which highly expresses the gene. Thepresent inventors further have found that 3-indole-pyruvic acid with ahigher yield is obtained by reacting tryptophan in the presence of aculture obtained by subjecting the transformant to a high densitycultivation or a treated culture, and completed the present invention.

Accordingly, the present invention provides the respective inventions asfollows.

[1] A polynucleotide selected from the group consisting of following (a)to (e):(a) a polynucleotide encoding a protein having the amino acid sequenceof SEQ ID NO:2;(b) a polynucleotide encoding a protein which has an amino acid sequencecomprising a substitution, deletion and/or insertion of one or severalamino acids in the amino acid sequence of SEQ ID NO:2 and has an oxidaseactivity;(c) a polynucleotide encoding a protein which has an amino acid sequencehaving 90% or more homology to the amino acid sequence of SEQ ID NO:2and has an oxidase activity;(d) a polynucleotide having the nucleotide sequence of nucleotidenumbers 61 to 1476 in the nucleotide sequence of SEQ ID NO:1; and(e) a polynucleotide which hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence of the nucleotide numbers 61 to1476 in the nucleotide sequence of SEQ ID NO:1 or with a probe capableof being prepared from said sequence under a stringent condition, andencodes a protein having an oxidase activity.[2] The polynucleotide according to the above described [1], whereinsaid stringent condition is a condition where washing is performed at asalt concentration corresponding to 1×SSC and 0.1% SDS at 60° C.[3] A recombinant polynucleotide having the polynucleotide according tothe above described [1] or [2].[4] A transformant introduced with the recombinant polynucleotideaccording to the above described [3].[5] A method for producing oxidase, characterized by culturing thetransformant according to the above described [4] in a medium toaccumulate the oxidase in the medium and/or the transformant.[6] A method for producing 3-indole-pyruvic acid, having a step ofconverting tryptophan into 3-indole-pyruvic acid in the presence of thetransformant according to the above described [4] and/or a culturethereof.[7] The method for producing 3-indole-pyruvic acid according to theabove described [6], wherein superoxide dismutase is added to a reactionsystem which converts tryptophan into 3-indole-pyruvic acid.[8] The method for producing 3-indole-pyruvic acid according to theabove described [6], wherein a transformant which expresses superoxidedismutase and/or a culture thereof is added to a reaction system whichconverts tryptophan into 3-indole-pyruvic acid.[9] The method for producing 3-indole-pyruvic acid according to theabove described [8], wherein said culture is obtained by rupturing cellmembrane of the transformant which expresses said superoxide dismutase.

Unless otherwise indicated herein, a sequence number indicates thesequence number described in Sequence Listing.

EFFECT OF THE INVENTION

According to the present invention, the polynucleotide encoding theprotein having the oxidase activity is provided. When such apolynucleotide is utilized, 3-indole-pyruvic acid that is the rawmaterial of monatin that is useful as the sweetener or the like can beproduced conveniently with the high yield by utilizing tryptophan, andit is industrially useful particularly in the field of foods.

BEST MODES FOR CARRYING OUT THE INVENTION 1. Polynucleotide According tothe Present Invention

First, the present invention provides a polynucleotide encoding aprotein having an oxidase activity.

In the present invention, oxidase means an enzyme that catalyzes anoxidative deamination of a substrate such as an amino acid, and havingthe oxidase activity means having an enzymatic activity of such oxidase.As representative ones of such a reaction, the reaction of convertingtryptophan into 3-indole-pyruvic acid (e.g., the reaction shown in thefollowing reaction formula 5 may be included) can be shown. The aminoacid such as tryptophan includes any of an L-form and a D-form, and D-and L-forms, but ordinary indicates the L-form.

In the present invention, preferably the oxidase catalyzes theaforementioned reaction and has a catalase activity. The catalaseactivity is the activity that catalyzes a decomposition reaction ofhydrogen peroxide, and for example, catalyzes the reaction shown in thefollowing reaction formula 6.

The polynucleotide of the present invention encodes the protein havingsuch an oxidase activity, and its representative may include (a) apolynucleotide encoding the protein having an amino acid sequence of SEQID NO:2.

The protein having the amino acid sequence of SEQ ID NO:2 has been newlyisolated from Providencia rettgeri AJ2770 strain and identified as theamino acid sequence of the protein having the oxidase activity by thepresent inventors. AJ2770 strain was internationally deposited toMinistry of International Trade and Industry, Agency of IndustrialScience and Technology, National Institute of Bioscience andHuman-Technology (currently Incorporated Administrative Agency, NationalInstitute of Advanced Industrial Science and Technology) on Nov. 28,1985, and deposit number FERM BP-941 was given thereto. AJ2770 strainwas originally identified as Proteus rettgeri, but as a result ofre-identification, it was classified into Providencia rettgeri(Providencia rettgeri sp).

A protein having the oxidase activity shown in the above reactionformula 5 can also be isolated from Proteus rettgeri IFO13501 strain.IFO13501 strain was deposited to Institute for Fermentation Osaka (17-85Juso-honmachi 2-chome, Yodogawa-ku, Osaka, Japan), but its business hasbeen transferred to NITE Biological Resource Center (NBRC) in Departmentof Biotechnology (DOB), National Institute of Technology and Evaluation(NITE) since Jun. 30, 2002, and the microorganisms are available fromNBRC with reference to the above IFO No.

The polynucleotide of the present invention includes polynucleotidesencoding substantially the same protein as the protein having the aminoacid sequence of SEQ ID NO:2. Specifically, the following polynucleotide(b) and polynucleotide (c) may be included:

(b) polynucleotide encoding a protein which has an amino acid sequencecomprising a substitution, deletion and/or insertion of one or severalamino acids in the amino acid sequence of SEQ ID NO:2 and has theoxidase activity; and

(c) polynucleotide encoding a protein which has an amino acid sequencehaving 90% or more homology to the amino acid sequence of SEQ ID NO:2and has the oxidase activity.

The “several amino acids” in the polynucleotide (b) vary depending onpositions and types of amino acid residues in a three-dimensionalstructure of the protein, are in the range in which thethree-dimensional structure and the activity of the protein with theamino acid residues are not significantly impaired, and specifically arepreferably 2 to 140, more preferably 2 to 95, more preferably 2 to 50,more preferably 2 to 30 and still more preferably 2 to 10 amino acidresidues. The sequence having mutations of one or several amino acidscan have the homology of 70% or more, preferably 80% or more, morepreferably 90% or more, still more preferably 95% or more and still morepreferably 98% or more to the sequence having no mutation. The homologyas used herein is a concept that represents a level of matching thesequences between two or more of the sequences. As one simple evaluationmethod of the homology, the number of the amino acid residues in a fulllength of the longer amino acid sequence in the two sequences is made adenominator, the number of the matched amino acid residues that mutuallycorrespond in the two sequences to be compared is made a numerator, andthis fraction can be calculated and multiplied by 100 to obtain thelevel expressed numerically. The homology can be calculated in greatdetail according to logic of bioinformatics, and various softwares havebeen developed for comparing similarity in the multiple sequences.Examples of the software for calculating the homology may include BLAST.

In the polynucleotide (c), the homology to the amino acid sequence ofSEQ ID NO:2 is preferably 70% or more, more preferably 80% or more, morepreferably 90% or more, more preferably 95% or more and still morepreferably 98% or more.

The polynucleotide (b) or the polynucleotide (c) is necessary to havethe oxidase activity. It is desirable that the enzyme encoded by thepolynucleotide retains the enzymatic activity at about a half or more,more preferably 80% or more and still more preferably 90% or more of theprotein having the amino acid sequence of SEQ ID NO:2 in a state havingno mutation, particularly under a condition at 30° C. at pH 8. Forexample, it is desirable to retain the enzymatic activity at about ahalf or more, more preferably 80% or more and still more preferably 90%or more of the protein having the amino acid sequence of SEQ ID NO:2,under the condition at 30° C. at pH 8.

The mutation of the amino acid as shown in the polynucleotide (b) isobtained, for example, by previously designing an amino acid sequencemodified so that an amino acid residue at a certain position in theamino acid sequence of SEQ ID NO:2 is substituted, deleted and/orinserted by site-specific mutagenesis, and expressing a nucleotidesequence corresponding to this amino acid sequence.

The mutation of the amino acid as shown in the polynucleotide (C) isalso obtained, for example, by previously designing an amino acidsequence modified so that the amino acid sequence in the certain regionof the amino acid sequence of SEQ ID NO:2 has the 90% or more homologyto the amino acid sequence of SEQ ID NO:2 by the site-specificmutagenesis, and expressing a nucleotide sequence corresponding to thisamino acid sequence.

The substitution, the deletion and/or the insertion of nucleotides asmentioned above includes naturally occurring mutations such asdifferences depending on species and strains of the microorganisms.Substantially the same protein as the protein having the amino acidsequence of SEQ ID NO:2 is obtained by expressing the polynucleotideencoding the amino acids having the mutation as mentioned above in anappropriate cell and examining the enzymatic activity of the expressedproduct.

Multiple nucleotide sequences defining each amino acid sequence can bepresent due to degeneracy of codons in the aforementionedpolynucleotides (a) to (c). The representative of specific examples ofthe polynucleotides (a) to (c) may include the following polynucleotide(d):

(d) polynucleotide having a nucleotide sequence of nucleotide numbers 61to 1476 in a nucleotide sequence of SEQ ID NO:1.

The polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1has been isolated from Providencia rettgeri AJ2770 strain. Thepolynucleotide consisting of the nucleotide sequence of nucleotidenumbers 61 to 1476 in SEQ ID NO:1 is a region of a coding sequence(CDS), and the amino acid sequence encoded by this CDS region is shownin SEQ ID NO:2.

Various gene recombination techniques mentioned below can be carried outin accordance with descriptions in Molecular Cloning, 2nd edition, ColdSpring Harbor press (1989) or the other references.

The polynucleotide (d) of the present invention can be obtained from themicroorganism having the oxidase activity, preferably the microorganismhaving the amino acid oxidase activity that acts upon tryptophan and thecatalase activity. For example, it can be acquired from chromosomal DNAor DNA library of Providencia rettgeri by PCR (polymerase chainreaction, see White, T. J. et al; Trends Genet., 5, 185, 1989) orhybridization. Primers used for PCR can be designed based on an internalamino acid sequence determined based on the oxidase purified fromProvidencia rettgeri. The primers and a probe for the hybridization canbe designed based on the nucleotide sequence of SEQ ID NO:1, and it canbe isolated using the probe. When the primers having the sequencescorresponding to 5′-untranslated region and 3′-untranslated region areused as the primers for PCR, the full length of the coding region of theenzyme can be amplified. Specifically, the 5′ side primer may includethe primer having the nucleotide sequence of an upstream region from thenucleotide number 61 in SEQ ID NO:1. The 3′ side primer may include theprimer having the sequence complementary to the nucleotide sequence of adownstream region from the nucleotide number 1476 in SEQ ID NO:1.

The primers can be synthesized, for example, using a DNA synthesizermodel 380B supplied from Applied Biosystems and using a phosphoamiditemethod (see Tetrahedron Letters, 1981, 22, 1859) according to standardmethods. PCR can be performed, for example, using Gene Amp PCR System9600 (supplied from Perkin Elmer) and TaKaRa LA PCR in vitro Cloning Kit(supplied from Takara Shuzo Co., Ltd.) according to the methodsdesignated by suppliers of respective manufacturers.

The polynucleotide of the present invention includes substantially thesame polynucleotide as the polynucleotide (d). Substantially the samepolynucleotide like this may include the following polynucleotide (e):

(e) polynucleotide which hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence of the nucleotide numbers 61 to1476 in the nucleotide sequence of SEQ ID NO:1 or with a probe capableof being prepared from the sequence under a stringent condition, andencodes a protein having an oxidase activity.

The “stringent condition” in the present invention refers to thecondition where a so-called specific hybrid is formed whereasnon-specific hybrid is not formed. Although it is difficult to clearlyquantify this condition, examples thereof may include the conditionwhere a pair of polynucleotides having high homology, e.g.,polynucleotides having the homology of 50% or more, more preferably 80%or more, still more preferably 90% or more, among others preferably 95%or more and still more preferably 97% or more are hybridized each otherwhereas a pair of polynucleotides having lower homology than that arenot hybridized each other, or a washing condition of an ordinarySouthern hybridization, i.e., hybridization at salt concentrationsequivalent to 1×SSC and 0.1% SDS at 60° C. and preferably 0.1×SSC and0.1% SDS at 60° C. The genes which hybridize under such a conditioninclude those in which a stop codon exists in the internal sequence oran activity is lost by the mutation of an active center. However, thosemay be easily removed by ligating the gene to the commercially availablevector, expressing it in the appropriate host, and measuring theenzymatic activity of the expressed product by the methods describedlater.

The probe in the polynucleotide (e) consists of the sequence capable ofbeing prepared from the sequence complementary to the nucleotidesequence in SEQ ID NO:1. Such a probe can be made by PCR with a DNAfragment containing the nucleotide sequence of SEQ ID NO:1 as a templateusing oligonucleotides made based on the nucleotide sequence of SEQ IDNO:1 as the primers. When the DNA fragment having the length of about300 by is used as the probe, the washing condition in the hybridizationmay include washing with 2×SSC and 0.1% SDS at 50° C.

The aforementioned polynucleotide (e) is necessary to have the oxidaseactivity. For example, it is desirable that the enzyme encoded by thepolynucleotide (e) retains the enzymatic activity at about a half ormore, more preferably 80% or more and still more preferably 90% or moreof the protein defined by the amino acid sequence encoded by theaforementioned polynucleotide (d), under the condition at 30° C. at pH8. Specifically, it is desirable to retain the enzymatic activity atabout a half or more, more preferably 80% or more and still morepreferably 90% or more of the protein encoded by the nucleotide sequenceof the nucleotide numbers 61 to 1476 in the nucleotide sequence of SEQID NO:1, under the condition at 30° C. at pH 8.

A way for obtaining the polynucleotide (e) is not particularly limited,and for example, the polynucleotide (e) can be obtained by isolating thepolynucleotide which hybridizes with the polynucleotide consisting ofthe nucleotide sequence complementary to CDS of SEQ ID NO:1 or with theprobe prepared from the same nucleotide sequence under the stringentcondition, and encodes the protein having the oxidase activity, from thepolynucleotides encoding the enzyme having the mutation or the cellhaving the polynucleotides.

The probe can be made based on the nucleotide sequence of SEQ ID NO:1according to the standard method. The method of isolating the objectivepolynucleotide by picking up the polynucleotide which is hybridized witha used probe may be performed according to the standard method. Forexample, a DNA probe can be prepared by amplifying the nucleotidesequence cloned into a plasmid or a phage vector, cutting out thenucleotide sequence to be used as the probe with restriction enzymes andextracting it. Sites to be cut out can be controlled depending on theobjective polynucleotide.

The polynucleotide modified like this can be acquired by conventionallyknown mutagenesis. Examples of the mutagenesis may include the method oftreating the polynucleotide encoding the enzyme, e.g., the abovepolynucleotide (d) with hydroxylamine or the like in vitro, and methodof treating bacteria having the polynucleotide encoding the enzyme andbelonging to genus Escherichia with ultraviolet light or a mutagen suchas N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid ordinaryused for the artificial mutagenesis.

2. Recombinant Polynucleotide and Transformant, and Method for ProducingOxidase According to the Present Invention

Second, the present invention provides a recombinant polynucleotidehaving the aforementioned polynucleotide of the present invention, atransformant introduced with the recombinant polynucleotide, and amethod for producing oxidase, which is characterized by culturing thetransformant in a medium to accumulate the oxidase in the medium and/orthe transformant.

As a host for expressing the protein specified by the polynucleotide,various prokaryotic cells including the genus Escherichia such asEscherichia coli and Bacillus subtilis, and various eukaryotic cellsincluding Saccharomyces cerevisiae, Pichia stipitis and Aspergillusoryzae can be used.

The recombinant polynucleotide used for introducing the polynucleotideinto the host can be prepared by inserting the polynucleotide to beintroduced into the vector corresponding to the type of the host in aform in which the protein encoded by the polynucleotide can beexpressed. As a promoter for expressing the polynucleotide of thepresent invention, if a promoter inherent in the oxidase gene inProvidencia rettgeri works in the host cell, the promoter can be used.If necessary, the other promoter may be ligated to the polynucleotide ofthe present invention to express the polynucleotide under the control ofthis promoter.

The method of transformation for introducing the recombinantpolynucleotide into the host cell may include D. M. Morrison's method(Methods in Enzymology 68, 326, 1979) or the method of enhancingpermeability of the polynucleotide by treating receiving microbial cellswith calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159,1970).

In the case of producing a protein on a large scale using therecombinant polynucleotide technology, a preferable embodiment mayinclude formation of an inclusion body of the protein, which is treatedto associate the protein in the transformant producing the protein. Theadvantages of this expression production method may include theprotection of the objective protein from digestion by proteases presentin the microbial cells, and ready purification of the objective proteinthat may be performed by disruption of the microbial cells and thefollowing centrifugation.

The protein inclusion body obtained in this way is solubilized by aprotein denaturing agent, which is then subjected to activationregeneration mainly by eliminating the denaturing agent, to be convertedinto the correctly refolded and physiologically active protein. Thereare many examples of such procedures, such as activity regeneration ofhuman interleukin 2 (JP 61-257931 A).

To obtain the active protein from the protein inclusion body, a seriesof the manipulations such as solubilization and activity regeneration isrequired, and thus the manipulations is more complicate than those inthe case of directly producing the active protein. However, when aprotein which affects microbial cell growth is produced on a large scalein the microbial cells, the affection can be avoided by accumulating theprotein as the inactive inclusion body in the microbial cells.

The methods for producing the objective protein on a large scale as theinclusion body may include methods of expressing the protein alone undercontrol of a strong promoter, and methods of expressing the objectiveprotein as a fusion protein with a protein known to be expressed in alarge amount.

The method for making transformed Escherichia coli (E. coli) andproducing the oxidase using this will be described more specificallybelow. When oxidase is produced in the microorganism such as E. coli, apolynucleotide encoding a precursor protein containing a leader sequencemay be incorporated or a polynucleotide encoding a mature proteincontaining no leader sequence may be incorporated as the coding sequenceof the protein. This can be appropriately selected depending on aproduction condition, a form and a use condition of the enzyme to beproduced.

As the promoter for expressing the polynucleotide encoding the oxidase,the promoter ordinary used for producing a xenogenic protein in E. colican be used, and examples thereof may include strong promoters such asT7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, andPR promoter and PL promoter of lambda phage. As the vector, pUC19,pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119,pMW118, pMW219 and pMW218 can be used. In addition, the vectors forphage polynucleotides can also be utilized. Further an expression vectorthat contains the promoter and can express the inserted polynucleotidesequence can also be used.

In order to produce the oxidase as a fusion protein inclusion body, afusion protein inclusion body may be achieved by ligating a geneencoding other protein, preferably a hydrophilic peptide to an upstreamor downstream of the oxidase gene. Such a gene encoding the otherprotein may be the one which increases an accumulation amount of thefusion protein and enhances solubility of the fusion protein after stepsof denaturing and regenerating, and example of the candidates mayinclude T7 gene 10, β-galactosidase gene, dehydrofolate reductase gene,interferon γ gene, interleukin-2 gene and prochymosin gene.

The ligation of these gene to the gene encoding the oxidase is performedso that reading frames of codons are matched. Such a ligation may beperformed by ligation at an appropriate restriction enzyme site, or byutilization of synthetic polynucleotide with appropriate sequence.

In order to augment a production amount, it is sometimes preferable toligate a terminator, i.e., a transcription termination sequence to thedownstream of the fusion protein gene. This terminator may include rrnBterminator, T7 terminator, fd phage terminator, T4 terminator,terminator of tetracycline resistant gene, terminator of Escherichiacoli trpA gene, and the like.

As a vector to introduce the gene encoding the oxidase or the fusionprotein of the oxidase and the other protein into E. coli, so-calledmultiple copying types are preferable, and includes plasmids having areplication origin derived from ColE1, such as pUC type plasmids, pBR322type plasmids or derivatives thereof. The “derivative” as used hereinmeans the plasmids modified by the substitution, deletion and/orinsertion of a nucleotide. The modification as used herein includesmutagenesis with the mutagen and UV irradiation, and modification suchas the natural mutagenesis.

It is preferred that the vector has a marker such as an ampicillinresistant gene for selecting the transformant. As such a plasmid,expression vectors carrying strong promoters are commercially available[pUC types (supplied from Takara Shuzo Co., Ltd.), pPROK types (suppliedfrom Clontech), pKK233-2 (supplied from Clontech) and the like].

The recombinant polynucleotide is obtained by ligating a polynucleotidefragment obtained by ligating the promoter, the gene encoding theoxidase or the fusion protein of the oxidase and the other protein andthe terminator in some cases in this order, to a vector polynucleotide.

When E. coli cells are transformed using the recombinant polynucleotideand cultured, the oxidase or the fusion protein of the oxidase and theother protein is expressed and produced. As the host to be transformed,the strain usually used for the expression of a xenogenic gene can beused, and Escherichia coli JM109 strain is preferable. The methods oftransforming and selecting the transformant are described in MolecularCloning, 2nd edition, Cold Spring Harbor press (1989) or the otherreferences.

When the fusion protein is expressed, the fusion protein may be composedso as to be able to cleave the oxidase therefrom using a restrictionprotease which recognizes a sequence such as the sequence of bloodcoagulation factor Xa or kallikrein, which is not present in theoxidase.

As the production media, the media usually used for culturing E. coli,such as M9-casamino acid medium and LB medium may be used. Cultureconditions and production induction conditions may be appropriatelyselected depending on types of the vector marker, the promoter, the hostbacterium and the like.

The oxidase or the fusion protein of the oxidase and the other proteinmay be recovered by the following method: when the oxidase or the fusionprotein of the oxidase is solubilized in the microbial cells, themicrobial cells may be collected and then disrupted or lysed, to obtaina crude enzyme solution. If necessary, the oxidase or the fusion proteinthereof can further be subjected to purification in accordance withordinary methods such as precipitation, filtration and columnchromatography. In this case, methods utilizing an antibody against theoxidase or the fusion protein can also be utilized.

In the case where the protein inclusion body is formed, this may besolubilized with a denaturing agent. The inclusion body may besolubilized together with the microbial cells. However, considering thefollowing purification process, it is preferable to remove the inclusionbody before solubilization and then solubilize the inclusion body.recovering the inclusion body from the microbial cells may be performedin accordance with conventionally and publicly known methods. Forexample, the microbial cells are broken, and the inclusion body isrecovered by centrifugation and the like. The denaturing agent thatsolubilizes the protein inclusion body may includeguanidine-hydrochloric acid (e.g., 6 M, pH 5 to 8), urea (e.g., 8 M),and the like.

As a result of removal of the denaturing agent by dialysis and the like,the protein is regenerated as having the activity. Dialysis solutionsused for the dialysis may include Tris-hydrochloric acid buffer,phosphate buffer and the like. The concentration may be 20 mM to 0.5 M,and pH may be 5 to 8.

It is preferred that the protein concentration utilized at aregeneration step is kept at about 500 μg/ml or less. In order toinhibit self-crosslinking of the regenerated oxidase, it is preferredthat dialysis temperature is kept at 5° C. or below. Methods forremoving the denaturing agent may include a dilution method and anultrafiltration method in addition to this dialysis method. Theregeneration of the activity can be expected by using any of thesemethods.

3. Method for Producing 3-Indole-Pyruvic Acid According to the PresentInvention

A method for producing 3-indole-pyruvic acid according to the presentinvention is characterized by having a step of converting tryptophaninto 3-indole-pyruvic acid in the presence of the transformant of thepresent invention and/or the culture thereof. According to theproduction method of the present invention, the oxidase can be producedconveniently in a large amount. Thus, 3-indole-pyruvic acid can also beproduced rapidly in the large amount.

Tryptophan used in the present invention may be any of an L-form and aD-form, and D- and L-forms, but it is desirable to employ the L-form interms of easy availability and price.

The transformant of the present invention is as already described above.The culture of the transformant means ones obtained by culturing thetransformant. Cultivation may be either a liquid cultivation or a solidcultivation, and the type of the medium is not particularly limited.Examples of the culture of the transformant may include the medium usedfor the cultivation, substances produced by the cultured transformantand mixtures thereof. These cultures may be treated for the purpose ofretrieving the enzyme, e.g., treated with sonication, glass beads,French press, lyophilization, a lytic enzyme, an organic solvent or asurfactant. These treated cultures may be purified by the standardmethod such as liquid chromatography and ammonium sulfate fractionation,and additionally may be included in a carrageenan gel or apolyacrylamide gel or immobilized on a membrane of polyether sulfone orregenerated cellulose.

An amount of the transformant or the culture thereof may be the amount(effective amount) that elicits an objective effect, and those skilledin the art easily determine this effective amount by a simplepreliminary experiment. For example, in the case of washed wet microbialcells, the effective amount can be 1 to 40 mg per 100 mL of the reactionsolution.

In the production method of the present invention, when theaforementioned culture of the transformant of the present invention isallowed to act upon tryptophan, the culture of the transformant can bebrought into contact with tryptophan. For example, the production methodmay include the method in which the transformant of the presentinvention is cultured in the medium to accumulate the oxidase in thetransformant and/or the medium and tryptophan is added to this medium,and the method in which tryptophan is added to the culture of thetransformant previously cultured for obtaining the oxidase. The additionof tryptophan is performed collectively, intermittently or continuouslyin the range (e.g., 0.1 to 10%) in which the objective reaction is notinhibited. When added, the substrate can be added in the state of anaqueous solution or a slurry. For the purpose of increasing a solubilityand facilitating the dispersion, the substrate may be mixed with theorganic solvent or the surfactant that has no effect on the reaction.

In the production method of the present invention, reaction conditionscan be determined appropriately. A reaction pH is ordinary 3 to 10 andpreferably 5 to 9. A reaction temperature is ordinary 10 to 60° C. andpreferably 20 to 40° C. A reaction period of time is ordinary 0.5 to 120hours and preferably 0.5 to 24 hours. If necessary, stirring may becarried out and the cultivation may be stationary cultivation.

Produced 3-indole-pyruvic acid is recovered by the standard method, andcan be purified if necessary. When it is necessary to prevent thedecomposition of the produced 3-indole-pyruvic acid after thecultivation, a degassing treatment or a deoxidization treatment can alsobe carried out.

3-Indole-pyruvic acid produced by the production method of the presentinvention [step (1) in the reaction formula 7] as shown in the followingreaction formula 7 is useful as a starting material of4-(indole-3-ylmethyl)-4-hydroxy-glutamic acid{3-(1-amino-1,3-dicarboxy-3-hydroxy-butane-4-yl)indole} [monatin] thatis useful as a sweetener. That is, a precursor keto acid (IHOG) issynthesized by aldol condensation of 3-indole-pyruvic acid and pyruvicacid [step (2) in the reaction formula 7], and IHOG can be aminated atthe 2-position in the presence of an enzyme, to synthesize monatin [step(3) in the reaction formula 7].

One preferable embodiment of the method for producing 3-indole-pyruvicacid according to the present invention may include the embodiment inwhich superoxide dismutase (hereinafter sometimes abbreviated as SOD) isadded to the reaction system. Produced 3-indole-pyruvic acid can beoxidized spontaneously to generate superoxide depending on thecondition. The generated superoxide decomposes 3-indole-pyruvic acid,and the decomposition of 3-indole-pyruvic acid rapidly progresses by aradical reaction in some cases. Thus, by adding SOD in the reactionsystem, the superoxide is eliminated in the reaction system and thedecomposition of 3-indole-pyruvic acid by the superoxide can beinhibited. In addition to SOD, the addition of a radical scavenger suchas mercaptoethanol can also prevent the decomposition of3-indole-pyruvic acid.

It has been speculated that when SOD is used, hydrogen peroxide isgenerated and 3-indole-pyruvic acid can be decomposed by hydrogenperoxide. However, unexpectedly, by the addition of SOD, the amount of3-indole-pyruvic acid present in the reaction system could be kept asdemonstrated in the following Example. Although its mechanism is notexactly clear, it is speculated that the superoxide has a strongerdecomposition action upon 3-indole-pyruvic acid than hydrogen peroxide,thus even when the amount of hydrogen peroxide is slightly increased, byadding SOD in the reaction system to eliminate the superoxide,3-indole-pyruvic acid can stably remain in the reaction system.

SOD is obtainable as a commercially available enzyme. SOD may be addedas the purified enzyme to the reaction system. A transformant thatexpresses SOD may be made and the transformant or a culture thereof maybe added to the reaction system. Nucleotide sequences of genes encodingSOD (sod gene), e.g., sodA gene, sodB gene and sodC gene have beenpublished on public databases. With reference to these databases, anucleic acid fragment encoding SOD can be amplified and the transformantcan be made using this.

When the transformant is made, the transformant transformed so as toexpress the amino acid oxidase may further be transformed so as toexpress SOD, or alternatively the respective transformants may be madeseparately and allowed to coexist in the reaction system. When the aminoacid oxidase and SOD are co-expressed in one transformant, the nucleicacid fragments encoding each protein may independently be incorporatedinto each plasmid, or alternatively both the nucleic acid fragments maytandemly incorporated into one plasmid.

SOD is present as a microbial intracellular enzyme accumulated in themicrobial cell in some cases. Thus, one preferable embodiment mayinclude the embodiment in which SOD is allowed to be released from aninside of the transformant. SOD may be released by rupturing themembrane of the transformant. For example, the transformant expressingSOD may be cultured to accumulate a sufficient amount of SOD in thetransformant, and the membrane of the transformant may be ruptured torelease SOD accumulated in the transformant from the inside of thetransformant. By rupturing the membrane of the transformant thatsupplies SOD, a contact probability of SOD and the superoxide canfurther be enhanced. As a result, the decomposition of 3-indole-pyruvicacid can be suppressed to enhance the yield of 3-indole-pyruvic acid.The rupture of the membrane may be breaking the cell membrane of thetransformant. The rupture of the membrane may include sonication andbacterial lysis using a solvent such as toluene, and the like. When themembrane is ruptured using toluene, toluene deactivates the amino acidoxidase in some cases. Thus, SOD is produced separately from theproduction of the amino acid oxidase and the reaction system using this,and SOD may be isolated and added to the reaction system.

EXAMPLES Example 1 Microorganism Producing 3-Indole-Pyruvic Acid (IPA)

A medium containing 3 g of ammonium sulfate, 1 g of monopotassiumphosphate, 3 g of dipotassium phosphate, 10 mg of iron sulfate, 10 mg ofmanganese sulfate, 10 g of yeast extract and 10 g of peptone in 1 liter(pH 7.0) was made, 50 mL of the medium was dispensed into a 500 mLSakaguchi flask, which was then sterilized at 120° C. for 20 minutes touse for the cultivation of microorganisms (medium 1). A slant agarmedium (agar 20 g/L) containing 18 g of usual broth in 1 liter wasprepared. One loopful of the microorganisms cultured on this slant agarmedium at 30° C. for 24 hours was inoculated and cultured with shakingat 30° C. at 120 reciprocations/min for 16 hours. After the cultivation,microbial cells were centrifuged and prepared as the wet microbialcells.

The microorganisms were added into 20 mM Tris-HCl buffer (pH 8.0)containing 10 g/L L-Trp so that a weight of the wet microbial cells was1% (w/v) in a total amount of 50 mL and then the reaction was performedat 30° C. for one hour. When the wet microbial cells of Providenciarettgeri were added, the production of 5.25 g/L 3-indole-pyruvic acid(IPA) was confirmed.

Example 2 Isolation of Amino Acid Oxidase (Deaminase) Enzyme GeneDerived from Providencia rettgeri

The isolation of an amino acid oxidase (deaminase) enzyme gene will bedescribed below. Providencia rettgeri AJ2770 strain was used as themicroorganism. Escherichia coli JM109 strain was used as a host for theisolation of the gene, and pUC118 was used as a vector.

Providencia rettgeri AJ2770 strain was cultured on the agar mediumcontaining 18 g/L of the usual broth at 30° C. for 24 hours. One loopfulof the microbial cells was inoculated in the 500 mL of Sakaguchi flaskin which 50 mL of the medium 1 had been placed, and cultured withshaking at 30° C.

The medium (50 mL) after the cultivation was centrifuged (8,000 rpm, 4°C., 10 minutes), and the microorganisms were collected. Chromosomal DNAwas acquired from this microbial cells using QIAGEN Genomic-tip System(Qiagen) based on the method of its instructions.

The chromosomal DNA (5 μg) prepared from Providencia rettgeri AJ2770strain was completely digested with BamHI. DNAs (2 kb to 10 kb) wereseparated on 0.8% agarose gel electrophoresis, purified using Gene CleanII Kit (supplied from Funakoshi Corporation), and dissolved in 10 μL ofTE (Tris-EDTA). 4.5 μL of 10 μL TE, pUC118 BamHI and BAP (supplied fromTakara Shuzo Co., Ltd.) were mixed and ligated using DNA Ligation KitVer. 2 (supplied from Takara Shuzo Co., Ltd.). 0.5 μL of this ligationreaction solution and 100 μL of competent cells of Escherichia coliJM109 (supplied from Toyobo Co., Ltd.) were mixed to transformEscherichia coli. The resulting transformant was applied on a solidmedium appropriate for the cultivation of E. coli to prepare achromosomal DNA library.

A colony formed on a fixed medium was inoculated in TB medium (24 g ofyeast extract, 12 g of peptone, 4 mL of glycerol, 1 g of monopotassiumphosphate and 3 g of dipotassium phosphate in 1 liter, pH 7.0) in a96-well plate and cultured at 30° C. overnight. 100 μL, of this culturedmicrobial medium was added to 100 μL of 20 mM Tris-HCl buffer (pH 8.0)containing 10 g/L L-Trp, and reacted with shaking at 30° C. at 120reciprocations/min. A strain that developed a red color was collected asa bacterial strain having an L-Trp deaminase activity.

Concerning an activity unit (amino acid oxidase activity) of the enzymeused in the present invention, the amount of the enzyme that converted 1μmole of L-Trp per one minute was defined as one unit (U) when 20 mMTris-HCl buffer (pH 8.0) containing 10 g/L L-Trp was reacted withshaking at 30° C. at 120 reciprocations/min.

The strain in which the amino acid oxidase (deaminase) activity had beenconfirmed by the colorimetric method was cultured at 37° C. for 16 hoursin a test tube in which 3 mL of the TB medium containing 50 mg/Lampicillin had been placed. The strain had 2.84 U of the amino acidoxidase (deaminase) activity per 1 mL of the medium. Therefore, thecloned gene was confirmed to be expressed in E. coli. This strain wasdesignated as pTB2 strain. No activity was detected in the transformantin which pUC118 alone had been introduced as a control.

The plasmid possessed by Escherichia coli JM109 was prepared from theabove bacterial strain confirmed to have the amino acid oxidase(deaminase) activity using QIAprep Spin Miniprep Kit (250) (suppliedfrom QIAGEN), and a nucleotide sequence of the inserted DNA fragment wasdetermined. A sequencing reaction was carried out using BigDyeTerminator v3.1 Cycle Sequencing Kit (supplied from Applied Biosystems)based on its instructions. The electrophoresis was carried out using3100 genetic analyzer (supplied from Applied Biosystems).

As a result, since an open reading frame encoding the protein having thehomology to the amino acid oxidase (deaminase) was present, the clonedgene was confirmed to be the gene encoding the amino acid oxidase(deaminase). The nucleotide sequence of the full length of the aminoacid oxidase (deaminase) gene and the amino acid sequence correspondingthereto were shown in SEQ ID NO:1. The homology of the obtained openreading frame was analyzed using BLASTP program. As a result, thehomology of 71% to amino acid deaminase derived from Proteus mirabilis(gb|AAA86752.1|) and the homology of 57% to amino acid deaminase derivedfrom Proteus vulgaris (EC 3.5.4.-) were calculated.

Example 3 Preparation of Ps_aad Expressing Strain

A promoter region of trp operon on the chromosomal DNA of Escherichiacoli W3110 was amplified by PCR using oligonucleotides shown in SEQ IDNO:3 and SEQ ID NO:4 as the primers. The resulting DNA fragment wasligated to pGEM-Teasy vector (supplied from Promega). E. coli JM109 wastransformed with this ligation solution, and a strain having anobjective plasmid in which the trp promoter had been inserted in adirection opposite to a direction of lac promoter was selected fromampicillin resistant strains. Then, a DNA fragment containing the trppromoter obtained by treating this plasmid with EcoO109I/EcoRI wasligated to pUC19 (supplied from Takara) treated with EcoO109I/EcoRI. E.coli JM109 was transformed with this ligation solution, and a strainhaving an objective plasmid was selected from ampicillin resistantstrains. Subsequently, a DNA fragment obtained by treating this plasmidwith HindIII/PvuII was ligated to a DNA fragment containing rrnBterminator obtained by treating pKK223-3 (supplied from AmershamPharmacia) with HindIII/HincII. E. coli JM109 was transformed with thisligation solution, and a strain having an objective plasmid was selectedfrom ampicillin resistant strains. The obtained plasmid was designatedas pTrp4.

An objective gene was amplified by PCR using the plasmid possessed bythe above strain confirmed to have the amino acid oxidase (deaminase)activity as the template and using the oligonucleotides shown in SEQ IDNO:5 and SEQ ID NO:6 as the primers. This DNA fragment was treated withNdeI/HindIII, and the resulting DNA fragment was ligated to pTrp4treated with NdeI/HindIII. E. coli JM109 was transformed with thisligation solution, and a strain having an objective plasmid was selectedfrom ampicillin resistant strains. This plasmid was designated aspTrP4-Ps_aad.

E. coli JM109 having pTrP4-Ps_aad was cultured on LB agar mediumcontaining 50 mg/L ampicillin at 37° C. for 16 hours. One loopful of theobtained microbial cells was inoculated into a 500 mL Sakaguchi flask inwhich 50 mL of the TB medium (containing 50 mg/L ampicillin) had beenplaced, and a main cultivation was performed at 37° C. for 16 hours.Since 1 mL of the medium showed 0.74 U of the amino acid oxidase(deaminase) activity, the cloned gene was confirmed to be expressed inE. coli. No activity was detected in the transformant in which pTrp4alone had been introduced as the control.

Example 4 Evaluation of L-Trp Oxidization Activity in Parent Strain andTransformant

Providencia rettgeri AJ2770 strain was cultured on the agar mediumcontaining 18 g/L of the usual broth at 30° C. for 24 hours. One loopfulof this microbial cell was inoculated into a 500 mL Sakaguchi flask inwhich 50 mL of the medium 1 had been placed, and cultured with shakingat 30° C. for 16 hours. The resulting cultured medium had 0.69 U of anL-Trp oxidization activity per 1 mL of the medium.

One loopful of pTB2 strain was inoculated into a 500 mL Sakaguchi flaskin which 50 mL of the TB medium containing 100 mg/L ampicillin had beenplaced, and cultured with shaking at 37° C. for 16 hours. The resultingcultured medium had 3.5 U of the L-Trp oxidization activity per 1 mL ofthe medium. It was confirmed that pTB2 strain had the L-Trp oxidizationactivity that was about 5 times higher than Providencia rettgeri AJ2770strain.

Example 5 Conversion Reaction from Trp to IPA

One loopful of pTB2 strain was inoculated into a 500 mL Sakaguchi flaskin which 50 mL of the TB medium containing 100 mg/L ampicillin had beenplaced, and cultured with shaking at 37° C. for 16 hours. 25 mL of theresulting cultured medium was inoculated into a 5000 mL Sakaguchi flaskin which 500 mL of the TB medium containing 100 mg/L ampicillin had beenplaced, and cultured with shaking at 37° C. for 16 hours. Each reactionsolution (300 mL) containing 100 mmol/L of L-Trp, 20 mM Tris-HCl and0.0025% disfoam GD-1,3-K (supplied from NOF Corporation) at pH 7.0 wasprepared so as to include 120 mL of the resulting cultured mediumtherein.

The reaction was performed using a 1 liter jar fermenter by stirring at100, 200, 300 or 400 rpm at 30° C. Air was ventilated at a flow rate of300 mL/minute. IPA contained in the reaction solution was quantified. Aconcentration and a reaction time when IPA had achieved the highestconcentration were confirmed, and the concentration was 74 mM (after 28hours) at 100 rpm, 85 mM (after 11 hours) at 200 rpm, 80 mM (after 4hours) at 300 rpm or 70 mM (after 2 hours) at 400 rpm.

Example 6 Recovery of IPA

One loopful of pTB2 strain was inoculated into a 500 mL Sakaguchi flaskin which 50 mL of the TB medium containing 100 mg/L ampicillin had beenplaced, and cultured with shaking at 37° C. for 16 hours. 25 mL of theresulting cultured medium was inoculated into a 5000 mL Sakaguchi flaskin which 500 mL of the TB medium containing 100 mg/L ampicillin had beenplaced, and cultured with shaking at 37° C. for 16 hours. Each reactionsolution (300 mL) containing 100 mmol/L of L-Trp and 0.0025% disfoamGD-113-K at pH 7.0 was prepared so as to include 120 mL of the resultingcultured medium therein.

The reaction was performed using a 500 mL four-necked flask by stirringat 400 rpm at 30° C. The air was ventilated at a flow rate of 300mL/minute. After reacting for 5.5 hours, the ventilation of the air wasstopped, and argon gas was ventilated. Subsequently, the reactionsolution was adjusted to pH 4.0 using 1 M sulfuric acid, and centrifugedto remove the microbial cells. 1 M sulfuric acid was added to theresulting supernatant to adjust pH to 2.0. The supernatant was stirredat 20° C. overnight to conduct neutralization crystallization. A crystalwas filtrated, washed with water and dried under reduced pressure toyield 3.81 g of IPA crystal with a purity of 90%. As described above,IPA could be recovered by the convenient method without utilizing acomplicate purification step using an ion exchange resin and the like.

Example 7 Evaluation of Addition Effect of Superoxide Dismutase (SOD) orMercaptoethanol as Stabilizing Factor for IPA

Each solution (1 mL) containing 10 mM IPA, 10 mM Tris-HCl and 1%acetonitrile was placed in a test tube, and shaken at 25° C. at 150reciprocations/min for 10 hours. Then, IPA contained in the solution wasquantified, and reduced to 2.6 mM.

SOD (1, 10 and 100 U/mL), catalase (100 U/mL), ascorbic acid (10 mL),tocopherol (10 mM), DTT (1 and 10 mM), sodium hydrosulfate (10 mM),mercaptoethanol (1 and 10%), ethanol (10%), methanol (10%), glycerol(10%), SDS (10 mM), Tween 20 (10%), Triton X-100 (10%), sodium boratebuffer (10 mM), Tiron (10 mM) or sodium thiosulfate (10 mM) wasindividually added to the above IPA solution, and shaken likewise. IPAwas quantified in each case. Results are shown in Table 1.

TABLE 1 Additive Concentration Residual IPA (mM) None 2.6 SOD 1 U/ml 8.8SOD 10 U/ml 10.5 SOD 100 U/ml 10.8 Catalase 100 U/ml 4.4 Ascorbic acid10 mM 3.0 Tocopherol 10 mM 2.7 DTT 1 mM 3.1 DTT 10 mM 0.74 Sodiumhydrosulfate 10 mM 3.2 Mercaptoethanol  1% 6.4 Mercaptoethanol 10% 9.1Ethanol 10% 2.8 Methanol 10% 3.3 Glycerol 10% 2.0 SDS 10 mM 4.3 Tween 2010% 3.9 Triton X-100 10% 3.7 sodium borate buffer 10 mM 1.5 Triton 10 mM0.50 Sodium thiosulfate 10 mM 2.4

Example 8 Conversion Reaction from Trp to IPA in the Case of Adding SOD

One loopful of pTB2 strain was inoculated into a 500 mL Sakaguchi flaskin which 50 mL of the TB medium containing 100 mg/L ampicillin had beenplaced, and cultured with shaking at 37° C. for 16 hours. A reactionsolution (1 mL) of 150 mmol/L L-Trp and 20 mM Tris-HCl at pH 7.0 wasprepared so as to include 0.4 mL of the resulting cultured mediumtherein. SOD (E. coli-MnSOD, Sigma) was added to the reaction solutionat a final concentration of 100 U/mL.

Each reaction solution (1 mL) was placed in a test tube, and shaken at25° C. at 150 reciprocations/min for 6 hours. The amount of IPA producedin the reaction solutions was 61 mM in the absence of SOD and 120 mM inthe presence of SOD.

Example 9 Construction of SOD-Expressing Strain

E. coli JM109 strain was cultured on the LB agar medium at 37° C. for 16hours. A DNA fragment containing a sodA gene was amplified by PCR usingthis microbial cells as the template. The sodA gene encodes SOD A (Mntype) and its sequence had been registered as accession number ofEG10953 on the public database such as ecogene (URL:http://ecogene.org/index.php). SD-Nde-sodA-f (SEQ ID NO:7) andsodA-Hind-r (SEQ ID NO:8) were used as the primers. The resulting DNAfragment was digested with NdeI and HindIII, and ligated to the vectorpSFN described in International Publication No. 2006/075486 Pamphlet(pSFN Sm_Aet in Examples, particularly see Examples 1, 6 and 12), whichwas digested with NdeI and HindIII similarly. E. coli JM109 strain wastransformed with this ligation solution and a strain having an objectiveplasmid was selected from ampicillin resistant strains. The obtainedplasmid was designated as pSFN-sodA.

Likewise, pSFN-sodB [SD-Nde-sodB-f (SEQ ID NO:9) and sodB-Hind-r (SEQ IDNO:10) were used as the primers] and pSFN-sodC [SD-Nde-sodC-f (SEQ IDNO:11) and sodC-Hind-r (SEQ ID NO:12) were used as the primers] wereconstructed. pSFN-sodB is the plasmid in which a DNA fragment containinga sodB gene has been incorporated. pSFN-sodC is the plasmid in which aDNA fragment containing a sodC gene has been incorporated. SOD A (Mntype) that is an expression product of the sodA gene, SOD B (Fe type)that is an expression product of the sodB gene and SOD C (Cu—Zn type)are isozymes. The sequences of the sodB gene and the sodC gene had beenregistered as the accession numbers EG10954 and EG13419, respectively onthe public database such as ecogene.

pSFN vector digested with NdeI and HindIII was ligated to a DNA fragmenthaving multicloning sites, which was obtained by digesting pTrp4 withNdeI and HindIII. E. coli JM109 strain was transformed with thisligation solution and a strain having an objective plasmid was selectedfrom ampicillin resistant strains. The obtained plasmid was designatedas pSFN-mcs.

Example 10 Measurement of SOD Activity of SOD-Expressing Strains

The constructed SOD-expressing plasmid, pSFN-sodA was introduced into E.coli JM109 strain, and one loopful of the transformant was inoculatedinto 50 mL of the TB medium containing 100 mg/L ampicillin, and culturedwith shaking at 37° C. for 16 hours to obtain a cultured medium.

Likewise, a cultured medium of a transformant carrying pSFN-sodB, acultured medium of a transformant carrying pSFN-sodC and a culturedmedium of a transformant carrying pSFN-mcs were obtained.

Microbial cells were collected from these cultured media, and suspendedin BugBuster master mix (Novagen) to prepare microbial cell extractsolutions. The SOD activity in the obtained microbial cell extractsolution was measured using SOD Assay Kit-WST (Dojindo) and the SODactivity per 1 mL of the cultured medium was calculated. SOD (E.coli-MnSOD, Sigma) was used as the standard. 0.01 mM CuCl₂ was added tothe microbial cell extract solution of the SOD C-expressing strain, andthe SOD activity was measured.

As a result, it was demonstrated that any of the SOD-expressing strainshad the higher SOD activity than pSFN-mcs strain. The results are shownin Table 2.

TABLE 2 Plasmid SOD activity (U/ml) pSFN-mcs 53 pSFN-sodA 189 pSFN-sodB874 pSFN-sodC 7700

Example 11 Conversion Reaction from Trp to IPA Using SOD-ExpressingStrain

A reaction solution (1 mL) of 200 mmol/L L-Trp and 20 mM Tris-HCl at pH7.0 was prepared so as to include 0.1 mL of the cultured medium of theSOD-expressing strain and 0.4 mL of the cultured medium of pTB2 strain.Toluene (1%) was added to the cultured medium of the SOD A-expressingstrain, which was mixed and stirred, and then added to the reactionsolution. Likewise, toluene (1%) was mixed and stirred with the culturedmedium of the SOD B-expressing strain, which was then added to thereaction solution. CuCl₂ was added to the cultured medium of the SODC-expressing strain at the final concentration of 0.01 mM and they werestirred, which was then added to the reaction solution. A reactionsolution containing no cultured medium of the SOD-expressing strain, anda reaction solution in which SOD (E. coli-MnSOD, Sigma) had been addedat final concentration of 100 U/mL were also prepared.

Each reaction solution (1 mL) was shaken at 25° C. at 140reciprocations/min for 6 hours using the test tube. IPA contained in theresulting reaction solution was quantified, and the highestconcentration of IPA that was 129 mM was confirmed when the culturedmedium of the SOD B-expressing strain treated with toluene had beenadded. The results are shown in Table 3.

TABLE 3 Additive IPA (mM) — 96 SOD (sigma) 123 Cultured medium ofSodA-expressing strain 100 Cultured medium of SodA-expressing strain +Toluene 109 Cultured medium of SodB-expressing strain 119 Culturedmedium of SodB-expressing strain + Toluene 129 Cultured medium ofSodC-expressing strain 98 Cultured medium of SodC-expressing strain +CuCl₂ 123

1. A polynucleotide selected from the group consisting of following (a)to (e): (a) a polynucleotide encoding a protein having the amino acidsequence of SEQ ID NO:2; (b) a polynucleotide encoding a protein whichhas an amino acid sequence comprising a substitution, deletion and/orinsertion of one or several amino acids in the amino acid sequence ofSEQ ID NO:2 and has an oxidase activity; (c) a polynucleotide encoding aprotein which has an amino acid sequence having 90% or more homology tothe amino acid sequence of SEQ ID NO:2 and has an oxidase activity; (d)a polynucleotide having the nucleotide sequence of nucleotide numbers 61to 1476 in the nucleotide sequence of SEQ ID NO:1; and (e) apolynucleotide which hybridizes with a nucleotide sequence complementaryto the nucleotide sequence of the nucleotide numbers 61 to 1476 in thenucleotide sequence of SEQ ID NO:1 or with a probe which can be preparedfrom said sequence under a stringent condition, and encodes a proteinhaving an oxidase activity.
 2. The polynucleotide according to claim 1,wherein said stringent condition is a condition where washing isperformed at a salt concentration corresponding to 1×SSC and 0.1% SDS at60° C.
 3. A recombinant polynucleotide having the polynucleotideaccording to claim
 1. 4. A transformant introduced with the recombinantpolynucleotide according to claim
 3. 5. A method for producing oxidase,characterized by culturing the transformant according to claim 4 in amedium to accumulate the oxidase in the medium and/or the transformant.6. A method for producing 3-indole-pyruvic acid, having a step ofconverting tryptophan into 3-indole-pyruvic acid in the presence of thetransformant according to claim 4 and/or a culture thereof.
 7. Themethod for producing 3-indole-pyruvic acid according to claim 6, whereinsuperoxide dismutase is added to a reaction system which convertstryptophan into 3-indole-pyruvic acid.
 8. The method for producing3-indole-pyruvic acid according to claim 6, wherein a transformant whichexpresses superoxide dismutase and/or a culture thereof is added to areaction system which converts tryptophan into 3-indole-pyruvic acid. 9.The method for producing 3-indole-pyruvic acid according to claim 8,wherein said culture is obtained by rupturing cell membrane of thetransformant which expresses said superoxide dismutase.
 10. Arecombinant polynucleotide having the polynucleotide according to claim2.
 11. A transformant introduced with the recombinant polynucleotideaccording to claim
 10. 12. A method for producing oxidase, characterizedby culturing the transformant according to claim 11 in a medium toaccumulate the oxidase in the medium and/or the transformant.
 13. Amethod for producing 3-indole-pyruvic acid, having a step of convertingtryptophan into 3-indole-pyruvic acid in the presence of thetransformant according to claim 11 and/or a culture thereof.
 14. Themethod for producing 3-indole-pyruvic acid according to claim 13,wherein superoxide dismutase is added to a reaction system whichconverts tryptophan into 3-indole-pyruvic acid.
 15. The method forproducing 3-indole-pyruvic acid according to claim 13, wherein atransformant which expresses superoxide dismutase and/or a culturethereof is added to a reaction system which converts tryptophan into3-indole-pyruvic acid.
 16. The method for producing 3-indole-pyruvicacid according to claim 15, wherein said culture is obtained byrupturing cell membrane of the transformant which expresses saidsuperoxide dismutase.